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water. Adjust pH to 6.5 with 1N NaOH. Transfer to 250 mL flask; bring to volume with lab water.
16.
Sucrase Solution
–
Measure 10 mL of maleate buffer in a graduated cylinder. Deliver to sucrase vial. Cap and swirl
gently to dissolve. Divide into ~450 μL aliquots and store frozen at -20 °C (each tube is enough for 2 samples) for up to
a year.
17.
Acetate Buffer
–
Combine 2.9 mL of glacial acetic acid with 450 mL of lab water. Adjust pH to 4.5 with 1N NaOH.
Bring total volume to 500 mL with lab water.
18.
Fructanase solution
–
Dissolve contents of fructanase vial in 22 mL of acetate buffer. Swirl gently to dissolve. Divide
into ~1 mL aliquots and store frozen at -20 °C for up to a year (each tube should contain enough for 9 samples).
Preparation of Standards and Spikes
1.
Fructose stock solution 100 μg/mL
–
Weigh 0.05 ± 0.0025 g of fructose reference material and transfer to a 500 mL
volumetric. Bring to volume with lab water.
2.
Glucoheptose Internal Standard
–
Weigh 0.1 ± 0.005g of glucoheptose and transfer to a 100 mL volumetric. Bring to
volume with lab water.
It is recommended to portion out aliquots of this solution to avoid freeze thaw cycles and
extend useful life of reagent.
Store frozen at -20 °C for up to 6 months.
3.
Working standards
–
Store frozen at -20 °C for up to 6 months.
Table 2-1
Working Standard Preparation
Fructose (μg/mL)
WS4 = 50 mL stock + 5 mL IS→
100 mL
50
WS3 = 25 mL stock +5 mL IS→ 100 mL
25
WS2 = 5 mL stock +5 mL IS→ 100 mL
5
WS1 = 5 mL Stock + 10 mL IS → 200 mL
2.5
To prepare WS:
1. Add fructose stock solution to each flask according to the procedure above
2. Add IS solution to each flask
3. Dilute to volume with lab water
4.
Fructan Spikes
–
For both the internal Abbott samples as well as the SPIFAN matrices fructan material was weighed and
an amount of product powder was added such as to achieve the target level of fructan desired. The combined fructan
material and product powder were then reconstituted using lab water. Aliquots of these solutions were then saved in
plastic centrifuge tubes and frozen until the day of use. Over the course of testing these solutions it became apparent that
a solubility problem was occurring for the samples spiked with the HP material. It appeared to be falling out of solution
after going through freeze/thaw cycles (Evidenced by testing a clear, dissolved solution of HP in lab water that was and
then frozen. After thawing, this aliquot displayed visible precipitate and was cloudy/milky in appearance).
Table 2-2
Abbott In-House Fructan Spike Overview
Spike Level
(nominal as-fed)
0.03% 0.3% 0.3% 0.3% 3% 3%
3%
Matrix
Lab water
and 9%
sucrose*
Product #4 Product #4 Product #4 Product #4 Product #4 Product #4
Fructan Type
scFOS,
Ingredion
scFOS,
Ingredion
Oligofructose,
Beneo Orafti
P95
Oligofructose
and Inulin,
Beneo Orafti
Synergy 1
scFOS,
Ingredion
Oligofructose,
Beneo Orafti
P95
Oligofructose
and Inulin,
Beneo Orafti
Synergy 1
*No additional matrix used in this spike to very precisely control the amount of sucrose in the samples.
Fos-04 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE